Efficacy assessment of a novel endolysin PlyAZ3aT for the treatment of ceftriaxone-resistant pneumococcal meningitis in an infant rat model

Background Treatment failure in pneumococcal meningitis due to antibiotic resistance is an increasing clinical challenge and alternatives to antibiotics warrant investigation. Phage-derived endolysins efficiently kill gram-positive bacteria including multi-drug resistant strains, making them attractive therapeutic candidates. The current study assessed the therapeutic potential of the novel endolysin PlyAZ3aT in an infant rat model of ceftriaxone-resistant pneumococcal meningitis. Methods Efficacy of PlyAZ3aT was assessed in a randomized, blinded and controlled experimental study in infant Wistar rats. Meningitis was induced by intracisternal infection with 5 x 107 CFU/ml of a ceftriaxone-resistant clinical strain of S. pneumoniae, serotype 19A. Seventeen hours post infection (hpi), animals were randomized into 3 treatment groups and received either (i) placebo (phosphate buffered saline [PBS], n = 8), (ii) 50 mg/kg vancomycin (n = 10) or (iii) 400 mg/kg PlyAZ3aT (n = 8) via intraperitoneal injection. Treatments were repeated after 12 h. Survival at 42 hpi was the primary outcome; bacterial loads in cerebrospinal fluid (CSF) and blood were secondary outcomes. Additionally, pharmacokinetics of PlyAZ3aT in serum and CSF was assessed. Results PlyAZ3aT did not improve survival compared to PBS, while survival for vancomycin treated animals was 70% which is a significant improvement when compared to PBS or PlyAZ3aT (p<0.05 each). PlyAZ3aT was not able to control the infection, reflected by the inability to reduce bacterial loads in the CSF, whereas Vancomycin sterilized the CSF and within 25 h. Pharmacokinetic studies indicated that PlyAZ3aT did not cross the blood brain barrier (BBB). In support, PlyAZ3aT showed a peak concentration of 785 μg/ml in serum 2 h after intraperitoneal injection but could not be detected in CSF. Conclusion In experimental pneumococcal meningitis, PlyAZ3aT failed to cure the infection due to an inability to reach the CSF. Optimization of the galenic formulation e.g. using liposomes might enable crossing of the BBB and improve treatment efficacy.


Introduction
The emergence and the spread of antimicrobial resistance in the last decades are major threats to human health. Alternative treatment strategies targeting antibiotic-resistant bacteria are urgently needed. One such approach is the use of endolysins (or lysins). Lysins are bacteriophage-derived enzymes with peptidoglycan hydrolase activity and are thereby able to degrade the bacterial cell wall. Recent in vitro and in vivo results suggest that endolysins might be promising antimicrobial agents. First, they rapidly lyse bacterial cells including those that are multi-drug resistant [1][2][3][4][5][6]. Second, they act very specifically, killing only strains from closely related species and do not affect off-target commensal bacteria, which makes them less likely to disturb the physiologic microflora [1,4,7]. Third, the frequency of resistance emergence seems to be low [8,9]. Finally, they appear safe; in 2 recent clinical trials, no detrimental effects were observed after topical [10] or intravenous [11] application in humans.
Streptococcus pneumoniae is an opportunistic human pathogen that can cause diseases like sinusitis, otitis media, pneumonia, septicemia, and meningitis. Meningitis due to pneumococci is associated with important morbidity and mortality. Despite the availability of appropriate antibiotic treatment, pneumococcal meningitis (PM) is associated with a high case-fatality rate of up to 37% in high-income countries [12,13]. By the end of the 20 th century, a concerning increase in antimicrobial resistance was recognized for S. pneumoniae [14]. This was mitigated by the implementation of the pneumococcal conjugate vaccines (PCV), which led to a concomitant decrease in the rate of invasive pneumococcal diseases (IPD) [15,16] and overall antimicrobial resistance [17][18][19]. Yet, as high as 30% of infections caused by S. pneumoniae remain resistant to at least one antibiotic [20], and cephalosporin-resistance in PM represents an emerging threat [21]. Additionally, serotype redistribution caused by vaccine selection pressure has been observed [22][23][24], leading to the risk of the re-emergence and spread of non-vaccine serotypes that are resistant to antibiotics.
The efficacy of different pneumococcal endolysins (e.g. Cpl-1 and Pal) has been previously assessed in experimental in vivo models for pneumonia [25], endocarditis [26], bacteremia [4,6], and otitis media [27]. In an animal model of PM, Cpl-1 reduced bacterial loads within the central nervous system (CNS) after a single intracisternal or intraperitoneal injection [28]. Overall, in principle endolysins seem attractive therapeutic candidates to treat pneumococcal infections.
So far, however, the translation of promising preclinical applications of endolysin into clinical trials has been strikingly slow. We adapted a well-established infant rat model of PM and evaluated the efficacy of a novel endolysin called PlyAZ3a T for the treatment of a ceftriaxoneresistant S. pneumoniae serotype 19A strain in a randomized, blinded, and controlled animal study. As PM affects in particular children under the age of 5 years, infant rats are especially well suited to mimic pediatric bacterial meningitis, since they reflect the developmental stage of this specific population.

Infecting bacterial strain
A ceftriaxone-resistant (in accordance with CLSI guidelines, MIC � 2 μg/ml as determined by E-test, [Biomérieux, Nürtingen, Germany]) clinical isolate of S. pneumoniae, serotype 19A (identified as SPn28) was used. Bacteria were routinely grown overnight at 37˚C in brain heart infusion broth (BHI, BD, USA), adjusted to an optical density (OD 570 ) of 0.1, and then further diluted tenfold in fresh and pre-warmed BHI medium. The subculture was grown for 2 h to reach logarithmic growth phase. Colony forming units (CFU) were determined on Columbia sheep blood agar (CSBA) plates.

Characterization of PlyAZ3a T
To assess similarities of PlyAZ3a T (GenBank: EMG32405.1) to the well-characterized pneumococcal endolysin Cpl-1 (GenBank: NP_044837.1) protein alignment was performed using BLASTp [32]. Killing efficacy of PlyAZ3a T was assessed on SPn28 and compared to vancomycin. SPn28 was grown to exponential phase and treated with either PlyAZ3a T (1 μg/ml, 100 μg/ ml or 200 μg/ml), vancomycin (1 μg/ml) or PBS. Bacteria were incubated statically at 37˚C with 5% CO 2 and CFU were quantified before treatment, and 1, 2, 4 and 6 h after treatment. Prior to quantification on agar plates, 10 μl of PlyAZ3a T -treated samples was added to 90 μl of NaCl 0.85% containing 10% w/v choline (Sigma Aldrich, Buchs, Switzerland) in order to block further activity of the enzyme. Six biological replicates were performed for each condition. The effect of temperature and pH on enzymatic activity of PlyAZ3a T was evaluated as previously described by Son et al. [33].

Assessment of toxicity and inflammation in primary astroglial cell culture
The use of animal for cell culture experiments was approved by the animal care and experimentation committee of the canton of Bern, Switzerland (license no. BE 6/20). Primary astroglial cells were isolated and processed from 2-day old infant Wistar rats (central animal facility of the Medical Faculty of the University of Bern, Switzerland) as previously described [34,35] with minor adaptations. After 11 days of cultivation, cells were seeded in 24-well plates (~250'000 cells/well) precoated with poly-L-ornithine (0.01 mg/ml in PBS, Buchs, Sigma-Aldrich). Three days later, astroglial cells were exposed for 24 h to (i) sterile PBS (pH 7.4), (ii) 10 μg/ml lipopolysaccharide (LPS) from E. coli 026:B6 (Sigma Aldrich, Switzerland), (iii) 100 μg/ml PlyAZ3aT or (iv) 2% Triton X-100 (Merck, Darmstadt, Germany). Production of NO 2 − was assessed in the supernatant of cell cultures as previously described [34,35]. Viability of cells was assessed using an XTT assay, according to manufacturer's instruction (Cell Proliferation Kit II, Sigma Aldrich, Buchs, Switzerland). The experiment was performed twice and in biological replicates of 18 for every condition. Changes in inducible nitric oxide synthase (iNOS) expression were also assessed by immunofluorescence. Cells were fixed with 4% paraformaldehyde (PFA, Merck, Switzerland) diluted in PBS for 10 min at room temperature, followed by three washing cycles with sterile PBS. Permeabilization was done with 0.1% Triton X -100 (TX-100, Merck, Zug, Switzerland) for 5 min, followed by a 1 h blocking with blocking buffer (0.01% TX-100 + 2% bovine serum albumin, dissolved in PBS). Primary antibodies consisted of anti-GFAP mouse antibodies for astrocytes (dilution 1:1000; ThermoFisher, Reinach, Switzerland) and anti-NCOA3 rabbit antibodies for iNOS (dilution 1:500; Amsbio, Abingdon, UK). Secondary antibodies were goat anti-mouse Cy3 for astrocytes (dilution 1:500; ABCAM, Switzerland) and donkey anti-mouse Cy3 for iNOS (dilution 1:500; ThermoFisher, Switzerland), dissolved in blocking buffer. Secondary antibodies were added after overnight incubation of the primary antibodies at 4˚C and three washing cycles with PBS and incubated for additional 2 h at room temperature. 4 0 ,6 0 -diamidino-2-phenylindole (DAPI, ThermoFisher, Reinach, Switzerland) was used to visualize cell nuclei. Images were generated using fluorescence microscopy (OLYMPUS CKX53, Olympus LS, Germany).

Infant rat model of pneumococcal meningitis
All animal experiments were approved by the animal care and experimentation committee of the canton of Bern, Switzerland (license no. BE 5/20). Animals had access to water and food ad libitum and were kept in rooms with controlled temperature (22 ± 2˚C) and natural light.
A well-established infant rat model for PM was used as previously described [34-38] and adapted to use the serotype 19A clinical isolate SPn28. SPn28 was passaged 3 times in infant rats before further investigations were performed.
Mixed-sex Wistar rats (outbred, 11-day old) and their dams were purchased from Charles River Laboratories (Sulzfeld, Germany). Bacteria in logarithmic growth phase were used as inoculum and were washed twice by centrifugation (3100xg, 10 min at 4˚C) and resuspended in ice cold sterile saline (NaCl 0.85%), followed by dilution to reach the desired OD 570 of 0.15.
Bacterial inoculation (10 μl containing 3.9 ± 0.25 x 10 7 CFU/ml SPn28) was performed by injection into the cisterna magna. This amount of inoculum was chosen according to the results of LD 90 experiments (S1 Fig). CSF sampling was performed by puncture of the cisterna magna using a 30-gauge needle and blood sampling by puncturing the facial vein with a 20-gauge needle and collection of 1 drop (approximately 50 μl). Clinical signs of meningitis arose at 17 hours post infection (hpi), and this correlated with quantitative analysis of bacterial titers in the CSF. This timepoint was chosen for the first treatment dose. Clinical scoring and weighing were performed at 17, 24 and 42 hpi as described previously [36]. Briefly, clinical score was determined as follows: 5 = healthy with normal behavior; 4 = turns upright within 5 s, weight loss and/or appearance of fur; 3 = turns upright within 30 s; 2 = inability to turn upright; 1 = marked diminished motor activity, coma. Animals were sacrificed upon reaching a score of � 2 to meet ethical requirements. At 42 hpi all remaining animals were sacrificed by intraperitoneal (i.p.) injection of pentobarbital (Esconarkon 1 , 150 mg/kg, Streuli Pharma AG, Switzerland) followed by perfusion with 4% PFA. Brains were collected for histomorphometric analysis of cortical damage and hippocampal apoptosis, which was performed as previously described [35,39,40].
Pharmacokinetic study. In order to assess pharmacokinetic properties of PlyAZ3a T , infected animals were treated with a single i.p. injection of either 100 mg/kg or 400 mg/kg b.w. PlyAZ3a T at 17 hpi. Animals were separated in 2 groups (n = 16 per group) with different sample withdrawal times, in order to avoid excessive sampling frequency from the same animals. CSF and blood samples were taken at 15 min, 4 h and 12 h for the first group and at 2 h, 6 h and 12 h for the second group. If animals reached a clinical score � 2, samples were taken after sacrifice and included into the analysis. Blood was collected in serum Microvettes 1 200 Z (Sarstedt, Nümbrecht, Germany).
Treatment study. The procedure for the randomized, blinded and controlled study of PM was as follows. CSF sampling was performed at 17 hpi to confirm meningitis and all animals were randomly allocated into 3 groups using GraphPad (https://www.graphpad.com/ quickcalcs/randomize1.cfm). Treatment groups were (i) placebo (sterile PBS), (ii) PlyAZ3a T 400 mg/kg b.w. and (iii) Vancomycin 50 mg/kg b.w. (Vancocin, in saline, Teva, Petach Tikwa, Israel). Treatments were applied twice daily (12-hour time interval) through an i.p. injection. All persons involved in animal treatment were blinded regarding treatment groups until the end of the experiment. The primary endpoint was survival at 42 hpi. Secondary endpoints were (i) CFU in CSF and blood at 6 and 25 h post treatment and (ii) extent of cortical necrosis and hippocampal apoptosis in histopathological analysis.
The volume of CSF recovered from each animal during sampling was restricted and ranged from 5 to 15 μl. This resulted into a limit of detection for CFU quantification of 1 x 10 3 CFU/ ml.
Animals displaying a clinical score of � 2 prior to the time of randomization were sacrificed immediately and excluded from the analysis. Similarly, animals without established meningitis at randomization, defined by bacterial titers in CSF <1 x 10 4 CFU/ml at 17 hpi, were excluded from the analysis.

Quantification of PlyAZ3a T in CSF and blood samples
The amount of PlyAZ3a T in CSF and serum samples was assessed by quantitative near infrared Western Blot (NIR-WB). Proteins in CSF or serum were separated using 10% SDS-polyacrylamide gels. Western blotting and immunostaining were performed with WesternBrightTM MCF and MCF-IR kit (Advansta, San Jose, USA) according to manufacturer's instructions. PlyAZ3a T was detected as a 41 kD band, by using a primary antibody (custom-produced PlyA-Z3a T -specific polyclonal rabbit antibody, David's Biotechnology, Regensburg, Germany). Fluorescence was measured using a Fusion FX6 EDGE System (Vilber Lourmat, Collégien, France) with an exposure time of 2 min. Quantification of PlyAZ3a T was performed by comparison to a standard curve of PlyAZ3a T ranging from 2-0.02 μg using FIJI (version 1.8). The limit of detection was 10 μg/ml.

Analysis of cytokines in cell culture supernatant
Inflammatory cytokines known to be upregulated in PM (IL-1β, IL-6 and TNF-α) were measured in cell supernatants and CSF samples using a magnetic multiplex assay (Rat Magnetic Luminex 1 Assay, R&D Systems, Bio-Techne) on a Bio-Plex 200 station (Bio-Rad Laboratory) as reported previously [35,41].

Plate lysis assay for endolysin resistance testing
Plate lysis assay was performed as described previously [42-44] to assess resistance and activity against various pneumococcal serotypes. Bacteria recovered from animals was grown to exponential phase and plated on a CSBA to produce uniform bacterial lawns. PlyAZ3a T (0.5 μg) was spotted onto the plate and incubated overnight. Bacteria were classified as susceptible to PlyAZ3a T if a clear lysis zone was observed.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 1 (San Diego, USA). Differences in survival were calculated using log-rank tests. Differences between means of non-normally distributed groups (cytokines, cortical necrosis, hippocampal apoptosis) were assessed using Kruskal-Wallis test with Dunn's multiple comparison. Differences in CFU reduction in CSF and blood of animals were calculated using Fisher's exact test. A p value < 0.05 was considered statistically significant.

PlyAZ3a T lyses ceftriaxone-resistant S. pneumoniae in vitro
PlyAZ3a T is a bacteriophage-derived endolysin found within the genome of S. tigurinus strain AZ3a T (Accession number GCA_000344275.1). Alignment of the amino acid sequence of PlyAZ3a T and Cpl-1 (up to now the most extensively characterized pneumococcal endolysin) displayed 80% similarity for the putative catalytic domain and 47% similarity for the putative cell-wall binding domain (Fig 1A), suggesting PlyAZ3a T might be active against S. pneumoniae. Indeed, PlyAZ3a T exhibit a broad-spectrum lytic activity against S. pneumoniae, lysing 18 distinct serotypes including both PCV 13 (3, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) and non-PCV 13 serotypes (11A, 15A, 17F, 20, 22F, 23B and 35B), as tested by plate lysis assay. No resistance to the endolysin activity could be detected among the tested isolates (n = 18). PlyA-Z3a T lysed S. pneumoniae efficiently (>50% of maximal efficacy) at temperatures ranging from 25-42˚C ( Fig 1B) and for pH ranging from 5-7. Optimal activity occurred at pH 6, while pH � 9 completely inactivated the enzyme (Fig 1B). In time-kill curve assays, PlyAZ3a T showed a dose-dependent killing of the ceftriaxone-resistant strain SPn28. The bacteria were not eradicated even after 6 h of exposure to the lysin at the highest concentration of 200 μg/ml (Fig 1C). In contrast, vancomycin displayed a bactericidal effect and reduced counts below the detection limit within the same time period (Fig 1C).

PlyAZ3a T failed to control ceftriaxone-resistant pneumococcal meningitis
In primary astroglial cell cultures, no direct toxicity of PlyAZ3a T was observed (S2A and S2B PlyAZ3a T was not superior to placebo in improving survival of animals with PM (p > 0.05, Fig 2A). In contrast, the standard-of-care antibiotic vancomycin significantly improved survival with a rate of 70% survival (p = 0.02 versus PBS and PlyAZ3a T , Fig 2A). PlyAZ3a T did not significantly reduce bacterial loads in the CSF and blood over the course of infection when compared to placebo (p > 0.05, Table 1). On the opposite, vancomycin-treated animals displayed a consistent reduction of bacterial loads in both CSF and blood within the first 6 h after start of therapy (CSF p = 0.0182 and blood p = 0.021 compared to placebo, Table 1

Reduced bioavailability of PlyAZ3a T in CSF
We further investigated whether the lack of efficacy of PlyAZ3a T in the experimental meningitis model was associated either with the emergence of endolysin resistance or with an altered pharmacokinetic profile. First, bacteria recovered from PlyAZ3a T treated animals (60 colonies in total) were tested for resistance and all remained susceptible to lysis. Second, PlyAZ3a T was quantified from the serum and CSF of infected animals receiving a single dose of either 100 mg/kg or 400 mg/kg b.w. PlyAZ3a T . PlyAZ3a T was detected in the serum (Fig 2B), peaked after 2 h of intraperitoneal administration (268 μg/ml for 100 mg/kg group and 785 μg/ml for 400 mg/kg group), and was detected up to 12 h in the 400 mg/kg BW group, albeit at low levels ( Fig 2C). In contrast, PlyAZ3a T was not detected in the CSF of any of the animals tested, at any time point (Fig 2B, limit of detection 10 μg/ml).

Discussion
Despite the introduction of the pneumococcal conjugate vaccine 13 (PCV13), S. pneumoniae serotype 19A remains a major causative of invasive pneumococcal disease and meningitis [22,45]. In addition, serotype 19A is among the major serotypes associated with penicillin or  CSF and (B) in blood. Statistical significance was determined using Fisher's exact test. Animals displaying bacterial loads below the limit of detection throughout the experiment or missing follow-up sampling were excluded from the analysis.

PLOS ONE
Efficacy of endolysin PlyAZ3a T in an infant rat model of ceftriaxone-resistant pneumococcal meningitis cephalosporin resistance [22]. Having a wider range of treatment alternatives to combat ceftriaxone-resistant pneumococci is of necessity. Herein, we investigated whether a novel phage lysine recently isolated from S. tigurinus strain AZ3a T [31] would be effective for the treatment of S. pneumoniae serotype 19A either in vitro or in vivo, in an experimental bacterial meningitis model in infant rats. In vitro, PlyAZ3a T showed a broad host-range and was able to lyse all tested S. pneumoniae serotypes independently from their antibiotic resistance profile. PlyA-Z3a T induced a quick and dose-dependent reduction of bacterial loads of the ceftriaxone-resistant serotype 19A. Furthermore, the endolysin was safe and did not induce any evident direct toxic effect in astroglial cell cultures. Only minor inflammatory reactions were observed, mostly related to residual endotoxin activity.
In vivo, PlyAZ3a T failed to improve survival, to reduce bacterial numbers in the CSF or to prevent systemic spreading of the infection in blood compared to placebo. On the other hand, vancomycin significantly increased survival to 70%, which is in accordance with survival rates observed in previous studies when using standard of care treatment in this animal model [34,35,38]. PlyAZ3a T showed an important pharmacokinetic limitation, as endolysin could not be detected in CSF of endolysin treated animals most likely as a result of its inability to cross the BBB or the low stability of the protein in this environment. A possible solution to bypass this limitation is the use of direct intracisternal application, which is not devoid of problems either, as shown in a previous investigation assessing the pneumococcal endolysin Cpl-1 in the similar experimental setting [28]. There, Cpl-1 exhibited a short half-life time of~16 min after direct intracisternal application, leading to bacterial regrowth within 4 h and prompting the necessity of repeated intracisternal injections, which on their part where harmful [28]. An intracisternal-sustained release might help to optimize the pharmacokinetic profile. In bacterial meningitis, such an opportunity might be found when an external ventricular drain (EVD) is available, allowing for a continuous delivery of endolysin directly to the CNS thereby circumventing the BBB. Alternatively, innovative galenic formulations (e.g. using liposomes) might improve the ability of the endolysin to cross the BBB allowing for an intravenous administration.
Taken together, our results highlighted the pitfalls and drawbacks of the application of PlyAZ3a T , and possibly endolysins in general, in bacterial meningitis. Despite a well described break down of the BBB in bacterial meningitis the BBB seems to remain largely impermeable to such phage proteins. As such, endolysins might be more suitable for the treatment of other bacterial infections as it was shown for Cpl-1, such as bacteremia [4,6], pneumonia [25] or endocarditis [26] where the endolysin can efficiently and readily reach the infection site.